1. Field of the Invention
The present invention relates to a pretreatment instrument of saliva and a pretreatment method of saliva, used for identification and quantitation of Streptococcus mutans as one of cariogenic bacteria in human saliva by the immunochromatographic method utilizing an antigen-antibody reaction.
2. Description of the Conventional Art
It is known that there is a close relation between the presence of Streptococcus mutans and the generation of dental caries in a human mouth. If the presence or absence of Streptococcus mutans and the amount thereof in the human mouth can be simply examined, it is possible to grasp an incidence risk and the present incidence status, resulting in giving benefits to an extremely large number of people.
Hitherto, there has been employed an examination utilizing an antigen-antibody reaction in examining bacteria. For example, the enzyme labeled antibody method is a method for effecting the identification and quantitation by means of a density of color generation using an enzyme. However, this method was required to use a special cleaning device and complicated and precise operations for dealing with an antibody and a sample and to introduce incubation for the enzymatic reaction. Further, the immunofluorescent antibody method is a method for labeling an antibody with a fluorescent dye and specifically dyeing an antigen reacted with the antibody. However, this method is not a general method because a fluorescence microscope is needed as an assaying device.
For these reasons, a number of methods have been proposed as the method for simply utilizing the antigen-antibody reaction. For example, the assaying method utilizing chromatography, as disclosed in U.S. Pat. Nos. 5,591,645, 4,855,240, 4,435,504 and 4,980,298 and Japanese Patent Laid-Open Nos. 145459/1986 and 160388/1994, is a method with excellent simplicity, by which the presence or absence of an antigen and the amount thereof can be known only by incorporating a collected body fluid into a test solution containing the antigen for the purpose of the identification and quantitation and infiltrating the mixture into an examination instrument. Such a method is generally called an immunochromatographic method. In this method, a specified antibody that attaches to only a target antigen (this antibody will be hereinafter referred to as “specific antibody”) is infiltrated into one end of a porous membrane (a pore diameter: several tens μm) such as nitrocellulose, and in the middle of the porous membrane, another specific antibody similarly attaching to only a specified antigen is infiltrated in a strip-like form and immobilized in the porous membrane. The specific antibody as infiltrated into the one end of the porous membrane is previously colored by particles of, e.g., gold colloid. When a sample solution is infiltrated on the one end of the porous membrane wherein this specific antibody is present, if an antigen reactive with the specific antibody is present in the sample solution, the antigen is coupled with the specific antibody and moves in the porous membrane by capillarity in a state having the colored particles attached thereto towards the side opposite to the one end at which the sample solution is infiltrated. On the way of the movement, when the antigen passes through a place where another specific antibody is immobilized in a strip-like form, the antigen is trapped by the specific antibody in the porous membrane, whereby a strip-like blotting appears on the porous membrane. Thus, it is possible to know that the target antigen is present in the sample along with the amount thereof.
If such technique were applied, it would appear to realize the identification and quantitation of Streptococcus mutans in the mouth. Actually, however, this technique has not yet been put into practical use because it involves the following problems. That is, in principle, the sample that can be used in the immunochromatographic method must be able to pass through the porous membrane by capillarity. However, since a major sample used for the examination of bacteria in the mouth, such as Streptococcus mutans, is saliva, a viscous substance present in the saliva, called as mucin, plugs the pores of the porous membrane. Also, the mucin functions to agglutinate epithelial attachment cells having peeled off from an oral mucous surface and existing in the saliva. Accordingly, these substances plug the pores of the porous membrane, so that the Streptococcus mutans cannot pass through the porous membrane.
In addition, there is present other problem than the mucin, which makes the assay of Streptococcus mutans difficult. That is, though the subjective Streptococcus mutans is a bacterium having a diameter of about 1 μm in a single state, because it is a streptococcus, it often forms a chain of 10 to 20 or more, which will be a possible factor for inhibiting the movement in the porous membrane. Moreover, the Streptococcus mutans often produces viscous glucan from sucrose in foods and causes vigorous agglutination. The chain and agglutination of the Streptococcus mutans cause not only clogging of the porous membrane but also reduction of the surface area of the streptococcus, thereby influencing the number of antigens present on the surface of the Streptococcus mutans, resulting in lowering in the precision of the assay.
Thus, we, the present inventors, previously proposed a pretreatment instrument of saliva, which during identification and quantitation of Streptococcus mutans as one of cariogenic bacteria in human saliva by the immunochromatographic method, dissolves mucin in the saliva by a simple method, thereby enabling not only to inhibit agglutination of epithelial attachment cells by the mucin but also to suppress agglutination of Streptococcus mutans (see Japanese Patent Application No. 2001-92769).
That is, the present inventors found the following matters: when the saliva is treated with an aqueous sodium hydroxide solution, the mucin and glucan in the saliva are dissolved, thereby inhibiting the agglutination of the epithelial attachment cells, or acting to an outer membrane of the Streptococcus mutans to enable to suppress the agglutination of the Streptococcus mutans; also, when the saliva is treated with a specific acidic aqueous solution, chaining of the Streptococcus mutans is inhibited, thereby enabling to suppress the agglutination of the Streptococcus mutans; and in addition, when the saliva is treated with a mixture of the aqueous sodium hydroxide solution or acidic aqueous solution and a specific surfactant, proteins present in the Streptococcus mutans are solubilized, whereby the Streptococcus mutans can smoothly pass through the porous membrane.
However, when using such a pretreatment instrument of saliva, in order to mix the saliva with the aqueous sodium hydroxide solution or the specific acidic aqueous solution, or the mixture of the aqueous sodium hydroxide solution or the specific acidic aqueous solution and the specific surfactant, efficiently within a short period of time such that the Streptococcus mutans can be identified and quantitated by the immunochromatographic method, it is necessary to use a mixing device having a strong mixing action such as an electromotive mixer. However, since the amount of the saliva collected from a subject for the examination is generally very small, a specific mixing device must be used. In the case where such a specific mixing device is not available, it is impossible to identify and quantitate the Streptococcus mutans. On the other hand, the mixing device must be cleaned up every time after it has been used once, leading to a very troublesome operation.